Ultramicropump

Briefly, mice were anesthetized with a mix of ketamine (75 mg/kg) and medetomidine (1 mg/kg, i.p.) or isoflurane (preferred) and fixed on a stereotaxic frame. Analgesia was provided by buprenorphine (8 μg/kg, s.c.). AAVs were injected unilaterally into the striatum (A/P +0.7 mm, M/L ± 1.8 mm, D/V −2.5 mm, relative to bregma) using a 10 μl Hamilton syringe at a rate of 0.25 μl/min, controlled by an Ultramicropump (World Precision Instruments). In all experiments, except where noted, for each hemisphere we administered 2 × 10⁹ genomic particles or sterile PBS. We assume that the efficiency of transduction is similar in all groups because infections spread throughout the striata (Fig. 1b, c). However, we note that different volumes were used to match viral titre between all samples (3 μl injections for ZF-KOX; 1.5 μl injections for PBS, GFP and mZF-KRAB). Mice were killed at 2, 4 or 6 weeks after the injections, for qRT-PCR and histological analyses. To minimise animal use, the data at 2 weeks were taken from a subset of mice from our previous study [14 (link)].

Stereotaxic injections were performed as described previously (H. Wang et al., 2021 (link)). AAV5-CMV-GFP and AAV5-CMV-Cre-GFP viruses were prepared by Gene Therapy Center Vector Core, the University of North Carolina at Chapel Hill (titer of 10¹² unit/ml). Glass pipettes (Drummond catalog #5000-1001-X10) were pulled with a micropipette puller (P1000, Sutter). Pipette tips were heat polished (Micro Forge MF-830, Narishige), with their diameters to be 30–50 μm. Two-month-old male ErbB4^f/f mice were anesthetized with ketamine and xylazine (100 and 10 mg/kg, respectively, i.p.) and head-fixed unto a stereotaxic device (David Kopf Instruments). The skull was exposed via a small incision and two small holes were drilled into the skull (bregma: AP: −0.70 mm, ML: ±0.20 mm). Glass pipettes were attached to a 2.5-μl Hamilton syringe and inserted into the brain (DV: −4.75); viruses were injected bilaterally (200 nl, each side) at 50 nl/min by an Ultra Micro Pump (World Precision Instruments). Syringes were remained in the brain for 10 min after injection before being removed. Surgical procedures were performed under antiseptic conditions. Injection sites were verified by postexperimental immunohistochemical analysis (see below).

Mice were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (10 mg/kg). After making a guide track through the conjunctiva and sclera at the superior temporal hemisphere using a 30-gauge needle, a hand-pulled glass micropipette was inserted into the mid-vitreal cavity. Rotenone (1 μl; 10 mM; Sigma-Aldrich) or vehicle (DMSO) was injected into the vitreal chamber at a rate of 100 nl/s using an Ultra Micro Pump (World Precision Instruments, Inc. Sarasota, USA). Patency was confirmed following needle removal. For sAPPα co-treatment experiments, a second intravitreal injection of recombinant human sAPPα (6 ng/μl, Sigma-Aldrich) or vehicle (phosphate-buffered saline; PBS) was performed 30 min after injection of Rotenone.

The hMSCs were injected into the spinal cord in accordance with previously described methods [7 (link)]. Briefly, on the day following surgery to induce SCI, mice were re-anesthetized via inhalation of sevoflurane. The animals were placed face-down, and a truncated 29G needle with a 5.0-μL glass syringe (Hamilton, Reno, NV) was inserted directly into the intervertebral spinal cord (D/V -1.0 mm) between T10 and T11. We trained investigators to insert the needle to the depth of the anterior horn of the spinal cord by the injection of dye. Then, the depth of needle insertion was standardized at -1.0 mm from surface of which did not leak the dye from the needle tract during infusion or from the ventral spinal cord. The hMSCs (5 × 10⁵ cells/μL, n = 40 mice) or HBSS (n = 26 mice) was infused at a rate of 0.5 μL/min with an Ultra Micro Pump (World Precision Instruments, Sarasota, FL). Following infusion, the needle was left in place for 1 min to facilitate the diffusion of the solution into the tissue. Previously, we demonstrated that the fate of hMSCs does not differ between immunocompetent and immunodeficient animals after ischemia [8 (link)]. Therefore, in the present study, we refrained from using immunosuppressant following cell implantation [10 (link)].

Single injections of the recombinant HB-GAM characterized previously (40 (link)) (1 mg/ml) or vehicle (1x PBS) were done directly to the spinal cord lesion immediately after the spinal cord hemisection. Microinjector (UltraMicroPump with SYS Micro4 Controler, World Precision Instruments, Inc., Sarasota, Florida) with Hamilton syringe and attached glass electrode was used to administer 7 μl of solution slowly within 4 min.

Mice were anesthetized with 1% pentobarbital sodium (35 mg/kg, i.p.) and placed in a stereotaxic frame (RWD Life Science). Viruses were injected using a glass micropipette connected to a 10-µL NanoFil syringe. The infusion rate was 80 nL/min controlled by an UltraMicroPump with a Micro4 controller (World Precision Instruments). At the end of injection, the micropipette remained in place for 10 min before withdrawal.
AAV-Ef1α-DIO-hM3Dq-eYFP or AAV-Ef1α-DIO-eYFP from BrainVTA (Wuhan, China, 600 nL, 3–8×10¹² vg/mL) was infused into the MSDB using the coordinates (in mm from bregma): A/P +0.98; M/L 0; D/V −4.8, −4.5, and −4.2. The mice were subjected to behavioral tests 4 weeks after virus injection. Brains were then sectioned to verify the infusion sites. Data were excluded if virus expression was misdirected.